The secretory vesicle protein synaptotagmin (Syt) has been proposed to function as a calcium sensor in regulated exocytosis and trigger membrane fusion via calcium-dependent interactions with phospholipids and the SNARE proteins syntaxin and SNAP25. The objective of this proposal is to determine the functional role of synaptotagmin in regulated exocytosis and to identify the biochemical properties responsible for its function. The specific aims include: to determine if synaptotagmin is the calcium sensor for regulated exocytosis in vivo; and to determine the regulatory role of Syt-SNARE interactions and Syt-phospholipid interactions in calcium-dependent exocytosis. A Syt-deficient PC12 cell line, which exhibits a loss of calcium-dependent exocytosis, will be utilized to test for the ability of Syt calcium binding, SNARE binding, and phospholipid binding mutants to rescue the loss of function. These studies will elucidate the mechanism by which Syt regulates exocytosis in a calcium dependent manner. Identifying Syt as the calcium-sensor for exocytosis and determining which properties of Syt are responsible for its regulatory function could lead to the development of drug strategies for the treatment of a number of endocrine and neural pathologies.